Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan 2009 - March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(2001)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Stability in the solvent was analytically proved.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Hsd Cpb:WU (SPF-bred)
- Source: Harlan-Nederlands, 5960 AD Horst, The Netherlands
- Age at study initiation: 12-14 weeks
- Weight at study initiation: 204 - 261 g (females); 421 - 545 g (males)
- Housing: Starting from gestation day 0 individually in Type IIIh Makrolon cages on low-dust wood shavings.
- Diet and water: ad libitum
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approximately 55
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
Administration volume: 5 mL/kg bw

PREPARATION OF DOSING SOLUTIONS: The formulations were prepared as needed taking into account the analytically determined stability.

VEHICLE
- Lutrol® (PEG 400).
- Justification for use and choice of vehicle (if other than water): The test item in the vehicle gave a solution which was analytically confirmed to be stable for at least 8 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Content checks of the active ingredient in samples with concentrations of 20, 60, and 200 mg/mL were carried out twice during the study.
For this purpose representative samples were dissolved with a mixture of acetonitrile/ aqueous 0.05 % formic acid (4:1) and subsequently quantified by reversed phase (C18) HPLC with UV-detection (DAD, wavelength 200 nm). Standard solutions of the authentic test item were used for calibration. The results of the content checks in samples with concentrations of 20, 60, and 200 mg/mL during the study showed no meaningful deviation of the active ingredient content from the nominal value.
Details on mating procedure:
The animals were mated by placing two females overnight into a Type IIIh cage together with one male rat. If sperm was detected in the vaginal smear taken on the morning following mating, this day was regarded as day 0 of gestation.
Duration of treatment / exposure:
Days 6 - 20 p.c.
Frequency of treatment:
once daily (between 06:00 and 12:00 CET)
Duration of test:
From study initiation date to end of in-life-phase 35 days.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected based on the results of a previous dose toleration study in rats with 1000 mg/kg, which revealed no treatment related findings.

Examinations

Maternal examinations:
CLINICAL EXAMINATIONS: Yes
- Time schedule: The females were inspected from days 0 to 21 p.c. twice daily (once daily only on weekends, on public holidays, and on day 21 p.c.), and all findings were recorded. Attention was paid to disturbances in the general condition of the rats (appearance, behavior), and any alterations concerning their excretory products.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the females was determined on day 0 p.c. and daily from day 6 to day 21 p.c. Corrected body weight gain was determined by subtracting the uterus weight on day 21 p.c. from the body weight gain from days 0 to 21 p.c.

FOOD CONSUMPTION: Yes
- The food intake of the animals was determined from the difference in weight between the food offered and the food not consumed for the following days of gestation: Days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18, and 18 -21.

WATER CONSUMPTION: Yes
- Water intake was assessed daily by visual estimation of the quantities left over and reported together with clinical findings.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Cesarean sections were performed on day 21 p.c. without knowledge of treatment groups.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of dead and live fetuses: Yes
- Other: individual weight and appearance of the placentas
Fetal examinations:
- Sex of live fetuses
- Individual weights of live fetuses
- External examinations: Yes: [findings in alive and dead fetuses are included]
- Soft tissue and head examinations : Yes: [evaluation of about half of alive fetuses per litter]
- Skeletal and cartilage abnormalities : Yes: [evaluation in about half of alive fetuses per litter]
Statistics:
Differences between the control and test item treated groups were considered to be significant when p < 0.05. Significant differences from the control group are indicated with * for p < 0.05 and ** for p < 0.01. Statistical evaluation was performed on an Alpha 800 5/500 computer using the following methods:
Analysis of Variance (ANOVA); in case of significance Dunnett's test for feed intakes, body weights, body weight gains, and corrected body weight gains, uterine weights, number of corpora lutea per female, number of implantations per female, number of live fetuses per female and as percentage of implantations per female, placental weights per female, fetal weights per female.
2 by N CHI2 test; in case of significant differences Fisher's exact test with Bonferroni correction for fertility rate, gestation rate, number of implantations per group, number of preimplantation losses per group, number of postimplantation losses, early resorptions, late resorptions or dead fetuses per group, number of live fetuses per group as percentage of implantations per group, number of male or female fetuses or fetuses with indeterminable sex per group, number of placentas with findings or litters with placental findings per group, number of fetuses or litters with external, visceral or skeletal findings, with malformations or with external or visceral deviations per group.
Kruskal-Wallis test and in case of significant differences Dunn's test for number of preimplantation losses per female, number of postimplantation losses, early resorptions, late resorptions or dead fetuses per female, number of male or female fetuses or fetuses with indeterminable sex per female, number of placentas with findings per female, number of fetuses with external or visceral findings, with malformations or with external or visceral deviations per female.
CHI2 test (correction according to yates) for number of fetuses or litters with cartilaginous tissue observations.
Indices:
gestation rate

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Appearance, behavior, and mortality were unaffected at dose levels up to and including 1000 mg/kg bw/day.
Absolute and corrected body weight gains were unaffected by treatment at dose levels up to and including 1000 mg/kg.
Food intake, water intake, and fecal and urinary excretions were unaffected by treatment at dose levels up to and including 1000 mg/kg.
No treatment related gross pathological findings occurred at dose levels up to and including 1000 mg/kg.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Remarks:
Maternal toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: No adverse findings in maternal animals up to and incl. 1000 mg/kg

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The gestation rate was unaffected by treatment at dose levels up to and including 1000 mg/kg.
Appearance and weights of placentas were unaffected by treatment at dose levels up to and including 1000 mg/kg.
Postimplantation loss and correspondingly the number of fetuses were unaffected by treatment at dose levels up to and including 1000 mg/kg.
Fetal sex distribution was unaffected by treatment at dose levels up to and including 1000 mg/kg.
The fetal weights were unaffected by treatment at dose levels up to and including 1000 mg/kg.
A treatment related effect on malformations was not evident at dose levels up to and including 1000 mg/kg.
A treatment related effect on external and visceral deviations was not evident at dose levels up to and including 1000 mg/kg.
A treatment related effect on skeletal deviations (retardations, variations, including cartilaginous tissue findings) was not evident at dose levels up to and including 1000 mg/kg.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: No substance-related findings in fetuses up to and incl. 1000 mg/kg (highest dose tested)

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Executive summary:

A developmental toxicity study with the substance was performed according to OECD TG 414. Twenty-five inseminated female Wistar rats per dose group were administered (gavage) daily from day 6 to 20 p.c. the test substance at doses of 0, 100, 300, and 1000 mg/kg bw in Lutrol®(PEG 400). The fetuses were delivered by cesarean section on day 21 of gestation. Investigation was performed on general tolerance of the test substance by the females as well as on its effect on intrauterine development.

Appearance, behavior, mortality, absolute and corrected body weight gains, food intake, water intake, and fecal and urinary excretions were unaffected at dose levels up to and including 1000 mg/kg bw.

No treatment related gross pathological findings occurred at dose levels up to and including 1000 mg/kg.

The gestation rate, appearance and weights of placentas, postimplantation loss and correspondingly the number of fetuses, fetal sex distribution, and fetal weights were unaffected by the treatment at dose levels up to and including 1000 mg/kg.

A treatment related effect on malformations, external, visceral, and skeletal (including cartilage) deviations was not evident at dose levels up to and including 1000 mg/kg.

Summarizing and evaluating all data investigated revealed a NOAEL of 1000 mg/kg bw for each maternal toxicity and developmental toxicity.